This invention relates to a novel cell growing medium and novel process for growing living cells.
Over the years there has been a substantial amount of effort directed to the problem of providing a versatile and efficacious culture media from reproducible, fully-characterized components. This "ideal" approach is frequently compromised with respect to the "fully characterized" nature of the components. For example, much work has been done (e.g. as described in U.S. Pat. Nos. 3,128,228 by J. Michl and 3,953,290 by K. Uthne) describing the use of serum of blood fractions in cell culture media. However, these fractions have some of the disadvantages of whole serum and other bulk protein sources. They are not as fully characterized as is desirable; that is, they will differ from batch to batch and introduce unknown variables in the media. Such variables can affect the growth of cells and can do so in wholly umpredictable ways.
Other workers have recognized the value of culture media which is free of serum and other bulk protein supplements which can interfere with cell replication. To date, some of these attempts have proved partially successful. Although the media developed in each case has serious limitations.
Thus, Torney et al in U.S. Pat. No. 3,887,930 and U.S. Pat. No. 4,055,460 describes a media containing a particulate resin which provides an undesirable substrate on which cells can grow and from which their removal can be difficult.
The work described by Bower, Arthur and Fine, Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum free media, in In Vitro (Pages 558-563, Volume 12, No. 8, 1976), utilizes a media containing considerable amounts of ethyl alcohol and lipids. The high volatility of ethyl alcohol (much higher than that of the acqueous media), which is used in this media to disperse the lipids in the acqueous solution, predisposes their media to serious problems. Gradual evaporation of the alcohol must be expected in the conditions to be found in cell culture incubators (about 37.degree. C. with a variable CO.sub.2 concentration in the atmosphere), causing their media to separate into a biphasic (lipid-acqueous) mixture. This type of mixture would not be appropriate for most cell culture work, nor would its exact composition be easily regulated. Besides, high concentration of alcohol, by itself, may have undesirable effects upon cell growth in many situations.
Other work, as described by S. T. Donta in "The growth of functional rat glial cells in a serumless medium." in Experimental Cell Research (Pages 119-124, Volume 82, 1973), has succeeded in adapting a single, specialized cell line to grow in a chemically defined media without bulk protein supplementation. However, this media has been unable to support the growth of other cell lines, unless used in combination with albumin. Thus, the media is not versatile unless supplemented with proteins which are not fully characterized.
Still another work, as described in U.S. Pat. No. 4,049,494, relates to processes for growing cells in serum-less media wherein the cells are from cell lines which have been specially adapted to function in the media. Such processes and media, of course, are lacking in broad utility. U.S. Pat. No. 4,072,565 describes the use of protamine zinc insulin in processes limited to short-terms by limitations inherent in the media selected.
Therefore, even after one decides on the ideal of providing a serum-free media, it is a further problem to provide a media with a combination of nutrients versatile with respect to a cell type that can be grown in it. Also, these nutrients, once identified, must be made available to the cells in a controlled fashion, without the introduction of highly volatile components of biphasic mixtures of variable composition. Furthermore, such a media must be free of particulate elements which would introduce unwarranted growth surfaces. Finally, this media should not be supplemented with partially refined bulk protein sources.